The present invention relates generally to thiazolindolinone compounds having utility as cyclin dependent kinase II inhibitors useful, inter alia, for preventing/reducing the severity of epithelial cytotoxic effects (e.g., alopecia, plantar-paimar syndrome, and mucositis) incident to chemotherapy and/or radiation therapy. The present invention also relates to the synthesis of such compounds, and to formulations containing such compounds as pharmacological compositions for preventing/reducing the severity of epithelial cytotoxic effects in a patient subjected to corresponding chemotherapy and/or radiation therapy treatment.
Protein kinases play a critical role in the control of cell growth and differentiation and are key mediators of cellular signals leading to the production of growth factors and cytokines. See, for example, Schlessinger and Ullrich, Neuron 1992, 9, 383. A partial, non-limiting, list of such kinases includes abl, ARaf, ATK, ATM, bcr-abl, Bik, BRaf, Brk, Btk, CDK1, CDK2, CDK3, CDK4, CDK5, CDK6, CDK7, CDK8, CDK9, cfms, c-fms, c-kit, c-met, cRaf1, CSF1R, CSK, c-src, EGFR, ErbB2, ErbB3, ErbB4, ERK, ERK1, ERK2, Fak, fes, FGFRI, FGFR2, FGFR3, FGFR4, FGFR5, Fgr, FLK4, Fps, Frk, Fyn, GSK, gsk3a, gsk3b, Hck, IGF-1R, IKK, IKK1, IKK2, IKK3, INS-R, Integrin-linkedkinase, Jak, JAK1, JAK2, JAK3, JNK, JNK, Lck, Lyn, MEK, MEK1, MEK2, p38, PDGFR, PIK, PKB1, PKB2, PKB3, PKC, PKCa, PKCb, PKCd, PKCe, PKCg, PKCI, PKCm, PKCz, PLK1, Polo-like kinase, PYK2, tie1, tie2, TrkA, TrkB, TrkC, UL13, UL97, VEGF-R1, VEGF-R2, Yes and Zap70. Protein kinases have been implicated as targets in central nervous system disorders such as Alzheimers (Mandelkow, E. M. et al. FEBS Lett. 1992, 314, 315. Sengupta, A. et al. Mol. Cell. Biochem. 1997, 167,99), pain sensation (Yashpal, K. J. Neurosci. 1995, 15, 3263-72), inflammatory disorders such as arthritis (Badger, J. Pharm. Exp. Ther. 1996, 279, 1453), psoriasis (Dvir, et al, J. Cell Biol. 1991, 113, 857), and chronic obstructive pulmonary disease, bone diseases such as osteoporosis (Tanaka et al, Nature, 1996, 383, 528), cancer (Hunter and Pines, Cell 1994, 79, 573), atherosclerosis (Hajjar and Pomerantz, FASEB J. 1992, 6, 2933), thrombosis (Salari, FEBS 1990, 263, 104), metabolic disorders such as diabetes (Borthwick, A.C. et al. Biochem. Biophys. Res. Commun. 1995, 210, 738), blood vessel proliferative disorders such as angiogenesis (Strawn et al Cancer Res. 1996, 56, 3540; Jackson et al J. Pharm. Exp. Ther. 1998, 284, 687), restenosis (Buchdunger et al, Proc, Nat Acad. Sci USA 1991, 92, 2258), autoimmune diseases and transplant rejection (Bolen and Brugge, Ann. Rev. Immunol. 1997, 15, 371) and infectious diseases such as viral (Littler, E.Nature 1992, 358, 160), and fungal infections (Lum, R. T. PCT Int. Appl., WO 9805335 A1 980212).
Chemotherapeutic techniques and radiation therapy techniques are well established in the treatment of neoplastic conditions of various types. As concomitant side-effects to the administration of chemotherapy and/or radiation therapy, the patient may experience severe host epithelial cell toxicity. The consequences of damage to the proliferating epithelium induced by chemotherapy frequently include hair loss (alopecia), plantar-palmar syndrome and mucositis; such side effects, especially mucositis, are also known to occur as a result of radiation therapy. These side-effect conditions may be of varying severity, depending on the type, dosages and dosing schedule of the respective chemotherapy and/or radiation therapy involved.
The present invention relates to the compound: 
in one or more of the variant forms hereinafter more fully described.
In one aspect, the present invention provides a novel approach to preventing/reducing the severity of epithelial cytotoxicity side-effects of chemotherapy and/or radiation therapy, in a subject receiving such therapy, by administering to the subject an effective amount of one or more of such variant forms of the above thiazoliridolinone compound, as hereinafter more fully described.
Epithelial cytotoxicity side-effects that can be preventedir educed in severity in this manner include alopecia, plantar-palmar syndrome and/or mucositis is induced by chemotherapy and/or radiation therapy.
Variant species of such thiazolindolinone compound 
include the various forms, which are described in the ensuing discussion and characterised with reference to their melting points, x-ray diffraction crystallographic spectra, solubilities in solvent media, and methods of synthesis.
The invention also relates in another aspect to methods for preparing various forms of such thiazolindolinone compound.
A further aspect of the invention relates to a pharmaceutical composition comprising (a) at least one of the aforementioned forms of such thiazolindolinone compound, in an amount that is effective in preventing/reducing the severity of epithelial cell toxicity side effects induced by the administration of chemotherapy and/or radiation therapy, and (b) one or more pharmaceutically acceptable carriers, excipients or diluents.
Other aspects, features and advantages of the invention will be more fully apparent from the ensuing disclosure and appended claims.
The present invention provides a thiazolindolinone compound having various forms that are useful as cyclin dependent kinase inhibitors.
The thiazolindotinone compound has the formula: 
and may be prepared in variant forms, Forms A, B and C, as described more fully hereinafter.
A generalized synthesis scheme (Scheme 1) is set out below for the thiazolindolinone compound of the invention, and is described more fully in the subsequent Examples herein. 
The resulting product of such synthesis, 4-{[7-oxo-6,7-dihydro-8H-[1,3]thiazolo[5,4-e]indol-8-ylidene)methyl]amino}N-(4-pyridinylmethyl)-benzene sulfonamide, can be recovered from the synthesis reaction system and thereafter submitted to recrystallization using a specific solvent species, to yield a corresponding desired form of the compound for ultimate use, as hereinafter described.
The resulting form of the compound may then be formulated as the active ingredient in a pharmaceutically acceptable composition, for topical or otherwise non-systemic administration to a subject (e.g., a mammalian subject such as a human subject), to prevent/reduce the severity of epithelial cytotoxicity side effects (e.g., alopecia, plantar-palmar syndrome, and/or mucocitis) induced by chemotherapy and/or radiation therapy being administered to the subject.
To prevent/reduce the severity of alopecia induced by chemotherapy and/or radiation therapy in a subject receiving such therapy, the thiazolindolinone compound of the invention in a suitable variant form is preferably administered topically to the corporeal locus that is susceptible to alopecia, such as the head (e.g., the scalp, eyebrow regions, beard and mustache areas, etc.).
For such topical application, the CDK2 inhibitor compound may be formulated in a topical administration formulation by combination of the compound with a selected pharmaceutically acceptable vehicle (carrier, diluent or excipient), with the amount of the compound being sufficient to achieve the prevention or reduction in severity of the alopecia side effect, when administered in accordance with an appropriately designed treatment protocol. The formulation can be in any useful dosage unit form for corresponding administration.
Formulations of the thiazolindolinone compound may be constituted and administered in any other suitable manner, such as in a liquid formulation for aerosolized spray administration to the head region of a subject susceptible to chemotherapy-induced alopecia, or by a dermal patch or dressing containing the CDK2 inhibitor formulation in a releasable form, for positioning on the head in contact with the area of susceptibility.
The formulation may alternatively be formulated as a lotion, salve, gel, foam, paste, oil, creme, or other suitable form, for administration to the appropriate corporeal locus, e.g., to the scalp or other area of the head for preventing/reducing the severity of alopecia, with initial administration being followed by massage, brushing, or toweling to distribute the formulation on the scalp evenly for uniformity of therapeutic effect.
As one example of compositions that may be used for topical adminstration of the CDK2 inhibitor agents of the invention to the corporeal locus of a subject receiving chemotherapy and/or radiation therapy, a formulation of the type described in Tata, S., et al., Relative Influence of Ethanol and Propylene Glycol Cosolvents on Deposition of Minoxidil into the Skin, Journal of Pharmaceutical Sciences, Vol. 83, No. 10, October 1994, pp. 1508-1510, may be employed, the entire disclosure of which is incorporated herein by reference. Such a formulation may comprise a 2% solution of the active ingredient in 60% ethanol, 20% propylene glycol and 20% water, for topical administration of the solution to the scalp.
Still other formulations that may be usefully employed for topical administration of the CDK2 inhibitor agents of the invention, include the formulations identified in U.S. Pat. Nos. 5,849,733; 5,807,698; 5,625,031; and 5,486,509, the disclosures of which are incorporated herein by reference in their entireties.
Additional illustrative examples of formulations that may be usefully employed for the topical administration of the CDK2 inhibitor agent of the invention include: the lipid based formulations described in Hoffman, R. M., et al., Liposomes Can Specifically Target Entrapped Melanin To Hair Follicles in Histocultured Skin, In Vivo Cell. Dev. Biol., Vol. 29A: 192-194, March 1993, and in Niemiec, S. M., et al., Influence of Nonionic Liposomal Composition on Topical Delivery of Peptide Drugs into Pilosebaceous Units: An in Vivo Study Using the Hamster Ear Model, Pharmaceutical Research, Vol . 12, No. 8, 1995, pp. 1184-1188; and the polymeric microsphere formulations described in Rolland, A., et al., Site-Specific Drug Delivery to Pilosebaceous Structures Using Polymeric Microspheres, Pharmaceutical Research, Vol. 10, No. 12, 1993, pp. 1738-1744.
The formulation may be constituted to provide an appropriate dose for a desired dosing schedule. The dosage and dosage schedule may be readily determined for a given subject, within the skill of the art, based on the character of the chemotherapy and/or radiation therapy being employed.
Analogous considerations apply to the formulation and administration of the CDK2 inhibitor for prevention/reduction in severity of plantar-palmar syndrome, involving topical administration to the areas of the hands and feet susceptible to the syndrome as a side-effect of chemotherapy and/or radiation therapy.
For preventing/reducing the severity of mucositis, the cyclin dependent kinase II inhibitor may be formulated in a suitable topical formulation for application to the oral cavity mucosa. Illustrative delivery systems for the cyclin dependent kinase II inhibitor of the invention, as used to combat mucositis, include the formulations and delivery techniques described in Cullinan U.S. Pat. No. 5,496,828 issued Mar. 5, 1996 for xe2x80x9cMethods of Inhibiting Ulcerative Mucositis.xe2x80x9d Useful formulations may include the active ingredient and excipients, diluents, or carriers, formed into tablets, capsules, sprays, mouthwashes, lozenges, troches, pastilles, lollipops, suspensions, powders and the like, for application to the mucosa of the oral cavity. cceptable daily dosages of the CDK2 inhibitor for preventing/reducing the severity of epithtelial cytotoxicity side effects induced by chemotherapy and/or radiation therapy, may be from about 0.1 to about 1000 mg/day, and preferably from about 50 to about 200 mg/day.
The cyclin dependent kinase II inhibitor in the preferred practice of the invention is administered contemporaneously with the chemotherapy and/or radiation therapy treatment (i.e., simultaneously with, or sufficiently near in time to, the chemotherapy and/or radiation therapy, so as to achieve a preventative or ameliorative effect on the therapy-induced epithelial cytotoxicity side effect that would otherwise be presented in the absence of the CDK2 inhibitor). The chemotherapy and/or radiation therapy may be of any appropriate type for the neoplastic condition, or other disease state or condition, of the patient being treated. As an illustrative example, the chemotherapy may comprise administration of chemotherapeutic agents, including cycle-specific agents (such as cytosine arabinoside (ARA-C)) and non-cycle-specific agents (such as Cytoxan), individually or in combination with one another.
The cyclin dependent kinase II inhibitor in one embodiment of the invention is administered 1-4 times in a chemotherapeutic cycle, as a cytoprotective composition for preventing/reducing the severity of epithelial cytotoxicity side effects such as alopecia, plantar-palmar syndrome and/or mucositis, in a subject receiving chemotherapy and/or radiation therapy.
In the specific application of preventing/reducing the severity of chemotherapy-induced alopecia, the cyclin dependent kinase II inhibitor effects a desired temporary arrest of the hair follicle cell cycle by inhibition of cyclin dependent kinase II activity. For such purpose, the inhibitor agent, formulated in a suitable topically administerable formulation, may be applied 1-2 times or more per chemotherapeutic cycle prior to and during the time of administration of chemotherapy, in one preferred specific embodiment.
In one preferred aspect of the invention, cyclin dependent kinase II inhibitor agents that are topically administerable to prevent/reduce the severity of chemotherapy-induced alopecia, are assessed for efficacy and selected for use based on the following characteristics:
(1) an IC50 value of less than 2.5 nanoMolar and preferably less than 20 nanoMolar against CDK2;
(2) an IC50 value of less than 1.5 microMolar and preferably less than 5 microMolar in a G1 checkpoint assay;
(3) exhibition of reproducible protection in a babyrat alopecia model using at least 2 different cytotoxic regimens one of which includes an alkylating agent (e.g., a regimen involving doxorubicin/cyclophosphamide (anthracyclin/alkylating agent), etoposide (topoisomerase II inhibitor), taxol, etc.);
(4) a topical dose of 10 mg/kg of body weight of the subject yielding a plasma concentration of less than 15 nanoMolar, and preferably a systemic exposure to less than 0.01 of the IC50 concentration for protection of the HT29 tumour cell line;
(5) an acceptable dermal irritation profile; and
(6) suitability for the particular topical formulation to be employed (in terms of compatibility, bioavailability, etc.).
The compound of the invention, in a suitable variant form, may correspondingly be formulated for topical administration to prevent/reduce the severity of plantar/palmar syndrome, in a suitable dosing and treatment regimen for the patient receiving chemotherapy and/or radiation therapy.
For the prevention/reduction of severity of mucositis in a patient receiving chemotherapy and/or radiation therapy, the compound of the invention in an appropriate form is preferably formulated for topical administration to the oral cavity mucosa, in a mouthwash, lozenge or lollipop.
The thiazolindblinone compound of the present invention has the ability to crystallize in more than one form, a characteristic known as polymorphism, and all such polymorphic forms (xe2x80x9cpolymorphsxe2x80x9d) are encompassed within the scope of the invention. Polymorphism generally can occur as a response to changes in temperature or pressure or both, and can also result from variations in the crystallization process. Polymorphs can be distinguished by various physical characteristics, and typically the x-ray diffraction patterns, solubility behavior, and melting point of the compound are used to distinguish polymorphs.
As used herein, the phrase xe2x80x9cin the vicinity ofxe2x80x9d in reference to a specified value of melting point temperature means a temperature (measured in degrees Centigrade) in the range of the specified value xc2x12xc2x0 C.
In the ensuing description, reagents employed in the synthesis of the invention are commercially available or are prepared according to procedures in the literature. The physical data given for the compounds exemplified is consistent with the assigned structure of those compounds. 1H NMR spectra were obtained on Varian Unity Plus NMR spectrophotometers at 300 or 400 Mhz. Mass spectra were obtained on Micromass Platform II mass spectrometers from Micromass Ltd., Altrincham, UK, using either atmospheric chemical ionization (APCI) or Electrospray Ionization (ESI). Analytical thin layer chromatography (TLC) was used to verify the purity of some intermediates which could not be isolated or which were too unstable for full characterisation, and to follow the progress of the reactions. Unless otherwise stated, this was done using silica gel (Merck Silica Gel 60 F254). Unless otherwise stated, column chromatography for the purification of compounds used Merck Silica Gel 60 (230-400 mesh), and the stated solvent system under pressure.
Melting points were determined for the respective forms of the thiazolindolinone compound of the invention by using differential scanning calorimetry with a TA model 2910. The sample mass was 1-3 mg in each instance. Each sample was contained in an aluminum crimped pan and was kept under a nitrogen purge at a flow rate of 40 milliliters/minute. The heating rate was 10xc2x0 C./minute.
Recrystallization procedures for the respective forms were carried out in each case with the compound being dissolved in the selected solvent and stored at ambient temperature (e.g., 25xc2x0 C.) until the solvent evaporated. The solid residue was then analyzed by x-ray diffraction.
In the respective anti-solvent determinations, the compound was dissolved in a selected solvent at 25xc2x0 C., and water or alcohol was added to precipitate a solid. The solid was analyzed by x-ray diffraction.
In the procedure for powder x-ray diffraction analysis, the powdered compound was sprinkled on a quartz zero background plate and scanned from 5-40xc2x0 2-Theta using a Scintag PADV or XDS2000.